The overall objectives for the studies proposed in this project is to delineate the mechanisms by which human Natural Killer cells are tolerant to normal autologous somatic cells; and define the conditions leading to NK cell activation by allogeneic cells. It has been proposed that NK cells in normal individuals achieve self-tolerance by endowing functional competence only to the inhibitory Killer immunoglobulin-like Receptors (KIR) for which they exhibit cognate HLA ligands. We hypothesize that engrafting NK cells following allogeneic hematopoietic stem cell transplantation (HCT) acquire tolerance to self through selective anergy of non-self inhibitory KIR. Prior to this acquisition of tolerance, donor NK alloreactivity due to missing KIR ligand in the host may be most evident. We also hypothesize that recognition of HLA class I by activating KIR only occurs when the HLA molecule is presented as an alloantigen,Jn contrast, when the HLA molecule is a self HLA molecule, the activating KIR is tolerized or unresponsive. These hypotheses will be tested in four specific aims: #1: Determine how NK alloreactivity is influenced by presence of activating KIRs KIR2DS2, 2DS1 and 3DS1 in individuals with different combinations of KIRhaplotypes andHLAclass I genotypes. This will beachieved bydetermining (a) if presence of the activating KIRs 2DS1, 2DS2 or 3DS1 correlates with a NK cell mediated alloresponse with specificity for the HLA-Cw and HLA-Bw4 KIR-ligand groups and (b) determine the functional NK phenotype in donors with the genes for the activating KIRs 2DS1, 2DS2 or 3DS1 and the corresponding KIR-HLA ligand group as self MHC antigen. #2: Determine if the development of NK cell alloreactivity and self tolerance associated with activating KIRs develops gradually over time during the period of engraftment. We will also examine if engrafting NK cells at any time display alloreactivity that deviates from donor NK alloreactivity. #3: (a) Determine acquisition of NK tolerance to self during'engraftment. We will determine if an initial period of hyper-responsiveness of inhibitory KIR is followed by selective anergy of non-self MHC-specific KIR. (b) Determine if the presence of T-cells in the allograft affects inhibitory KIR-mediated NK response. #4: Determine the acquisition of NK receptors to HLA class I during NK development in vitro. Wewill investigate NK development ex vivo from CD34 cells obtained from Cord Blood and other sources. We will determine the sequential acquisition of NK receptors, and differential acquisition of receptors within the same receptor family; determine their acquisition of function, tolerance to self and allogeneic reactivity. We will determine if these developmental patterns are affected by the HLA class I phenotype expressed in the stromal culture environment. Relevance: These studies will facilitate development of new methods for donor selection for patients with leukemia and for treatment with NK cell infusions post transplantation to enhance engraftment and leukemia resistance.